1. Field of the Invention
The present invention relates to combinations of monoclonal antibodies which specifically bind or recognize the antigen hepatocyte growth factor/scatter factor (HGF/SF) and inhibit HGF/SF activity. The application also relates to the use of the antibody in detection methods, in methods of identifying developmental disorders, and in therapy of particular conditions, such as cancer and liver disorders.
2. Background Art
HGF/SF is a heterodimeric molecule composed of an α-chain containing the N-terminal domain and four kringle domains (NK4), covalently disulfide linked to a serine protease-like β-chain. Acting through its receptor c-MET, HGF/SF initiates mitogenic, motogenic and morphogenic activities in a wide variety of cells. The N-terminal domain with the first kringle (NK1) or first two kringles (NK2) can bind to c-MET receptor with reduced affinity (10 and 4 fold respectively) when compared to wild-type HGF/SF, and both NK molecules show partial biological activity of HGF/SF (e.g., NK1 is an agonist while NK2 is an antagonist). The β-chain binds to the c-MET receptor specifically occupied with α-chain, but deletion of the β-chain results in loss of multiple biological activities of HGF/SF (1-5).
HGF/SF (hepatocyte growth factor/scatter factor) is an effector of cells expressing the Met tyrosine kinase receptor (Gherardi et al. 1993. Hepatocyte growth factor/scatter factor (HGF/SF), the c-met receptor and the behavior of epithelial cells.” Symp. Soc. Exp. Biol. 47:163-181; Matsumoto et al. 1992. “Hepatocyte growth factor: molecular structure, roles in liver regeneration, and other biological functions.” Crit. Rev. Oncog. 3:27-54 and Rubin et al. 1991. “Hepatocyte growth factor/scatter factor and its receptor, the c-met proto-oncogene product.” Biochim. Biophys. Acta 1155: 357-371). It is produced by mesenchymal cells and acts predominantly on cells of epithelial origin in an endocrine and/or paracrine fashion (Sonnenberg et al. 1993. “Scatter factor/hepatocyte growth factor and its receptor the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development.” J. Cell Biol. 123:223-235 and Stoker et al. 1987. “Scatter factor is a fibroblast-derived modulator of epithelial cell mobility.” Nature 327: 239-242). As its name implies, HGF/SF promotes the growth and/or scattering of various cell types. HGF/SF has also been shown to mediate other biological activities, including the formation of tubules (Montesano et al. 1991. “Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.” Cell 67:901-908) and lumens (Tsarfaty et al. 1992. “The met proto-oncogene receptor and lumen formation.” Science 257:1258-1261), the promotion of angiogenesis (Bussolino et al. 1992. “Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.” J. Cell. Biol. 119: 629-641), the inhibition of cell growth (Higashio et al. 1993. “Tumor cytotoxic activity of HGF/SF.” Exper. Suppl. 65:351-368) and the conversion from a mesenchymal to an epithelial phenotype (Tsarfaty et al. 1994. “Met mediated signaling in mesenchymal to epithelial cell conversion.” Science 263:98-101). In vivo, this ligand-receptor pair is believed to play a role in neural induction (Streit et al. 1995. “A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation.” Development 121:813-824), kidney development (Santos et al. 1994. “Involvement of hepatocyte growth factor in kidney development.” Dev. Biol. 163:525-529), tissue regeneration (Matsumoto et al. 1993. “Roles of HGF as a pleiotropic factor in organ regeneration.” Birkhauser-Verlag, Basel), wound healing (Nusrat et al. 1994. “Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium.” J. Clin. Invest. 93:2056-2065) and is required for normal embryological development (Uehara et al. 1995. “Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor.” Nature 373:702-705). Decreased levels of HGF/SF could result in defective organogenesis resulting in developmental abnormalities. Conversely, increased HGF/SF-Met signaling after tissue injury, such as hepatic injury, could lead to abnormal tissue regeneration which contributes to chronic hepatitis, cirrhosis, and/or liver cancer.
HGF/SF-Met signaling is also important in tumor development and progression. Met was originally isolated as the product of a human oncogene, trp-met, which encodes an altered Met protein possessing constitutive, ligand-independent tyrosine kinase activity and transforming ability. (Cooper et al. 1984. “Molecular cloning of a new transforming gene from a chemically transformed human cell line” Nature 311:29-33). The coexpression of unaltered Met and HGF/SF molecules in the same cell, which generates an autocrine stimulatory loop, induces an oncogenic transformation of those cells. (Bellusci et al. 1994. “Creation of a hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity.” Oncogene 9:1091-1099).
In addition to transforming cells, deregulated Met signaling in cells increases their invasiveness in vitro (Giordano et al. 1993. “Transfer of mitogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene.” Proc. Natl. Acad. Sci. USA 90:649-653) and metastatic potential in vivo (Rong et al. 1994. “Invasiveness and metastasis of NIH/3T3 cells induced by Met-HGF/SF autocrine stimulation.” Proc. Natl. Acad. Sci. USA 91:4731-4735). HGF/SF-Met signaling also induces the invasiveness and metastatic potential of other cell types (Bellusci et al. 1994). The detection of significant levels of HGF/SF in the pleural effusion fluid of patients whose cancer had metastasized to the pleura (Kenworthy et al. 1992. “The presence of scatter factor in patients with metastatic spread to the pleura.” Br. J. Cancer 66:243-247) demonstrates the involvement of HGF/SF-Met signaling in promoting metastasis in humans.
For example, although HGF/SF is synthesized by mesenchymal cells and acts predominantly on Met-expressing epithelial cells, it has been demonstrated that human sarcoma cell lines often inappropriately express high levels of Met and respond mitogenically to HGF/SF (Rong et al. 1995. “Met proto-oncogene product is overexpressed in tumors of p53-deficient mice and tumors of Li-Fraumeni patients. Cancer Res. 55:1963-1970 and Rong et al. 1993. “Met expression and sarcoma tumorigenicity.” Cancer Res. 53:5355-5360). It has also been shown that clinical sarcoma samples may overexpress the Met receptor (Rong et al. 1993 and Rong et al. 1995). Studies on lung adenocarcinomas revealed increased Met staining in these tumors (Ichimura et al. 1996. “Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance. Jpn. J. Cancer Res. 87:1063-1069 and Liu and Tsao 1993. “Proto-oncogene and growth factor/receptor expression in the establishment of primary human non-small cell lung carcinoma cell lines. Am. J. Pathol. 142:413-423.). Thus, this receptor-ligand pair is known to be involved in human oncogenesis and HGF/SF-Met signaling dramatically induces the in vitro invasiveness and in vivo metastatic potential of cells.
Due to its involvement in tumorigenesis, organogenesis, and regeneration, it is desirable to quantify the amount of HGF present in tissues in order to diagnose abnormalities associated with irregular HGF expression.
This invention provides monoclonal antibodies that allow characterization of HGF expression in tumor tissues. The diagnostic aspects of these antibodies can be extended to the characterization of developmental abnormalities associated with HGF/SF-Met signaling such as disorders of the kidney, liver, lung, skeletal muscle and other organs. In addition to its diagnostic properties, the HGF monoclonal antibodies act as inhibitors by blocking the interaction between HGF/SF and Met. In so doing, the antibody inhibits metastasis and decreases tumorigenecity. With respect to regenerative responses, the antibody is used to prevent abnormal regeneration and its untoward effects including tumorigenesis.
This invention provides several combinations of monoclonal antibodies that act as inhibitors of the MET-HGF/SF signaling pathway. In addition, these antibodies are useful in ELISA, immunoprecipitation studies and for immunohistochemical staining of paraffin sections. Since HGF/SF is known to be an important mediator of mitogenesis (hepatocytes), motogenesis (cell motility) and morphogenesis, and is involved in embryonic development, wound-healing, tissue organ regeneration, angiogenesis and carcinogenesis (7-10), these neutralizing Mabs are useful for a broad range of bio-medical research activities and clinical applications.